Construction and characterization of monoclonal antibodies against western equine encephalitis virus

ABSTRACT

Construction and characterization of mouse monoclonal antibodies against western equine encephalitis virus (WEE) for potential use in detection, diagnosis, and immunotherapy are disclosed. Antibodies were prepared from hybridoma cells and further characterized by ELISAs, Western blotting, isotyping, and immunoprecipitation. The antibodies were also tested for cross-reactivity to other alphaviruses, such as Sindbis virus (SIN), Venezuelan equine encephalitis virus (VEE), and eastern equine encephalitis (EEE). All antibodies bound to WEE antigen in ELISAs, whereas only a subgroup of antibodies was found to be active in Western blotting and immunoprecipitations. A subset of antibodies was found to cross-react with other alphaviruses, such as SIN, VEE, and EEE.

[0001] The present application is a divisional of U.S. Ser. No.09/793,606, filed Feb. 27, 2001.

FIELD OF THE INVENTION

[0002] This invention relates to the construction and characterizationof mouse monoclonal antibodies against western equine encephalitis virus(WEE) expressed from hybridoma cell lines.

BACKGROUND OF THE INVENTION LIST OF PRIOR ART LITERATURES

[0003] Cao Y and Suresh M R, Bispecific antibodies as novelbioconjugates. Bioconjug Chem 1998;9:635-644.

[0004] Boere W A M, Benaissa-Trouw B J, Harmsen M, Kraaijeveld C A, andSnippe H, Neutralizing and non-neutralizing monoclonal antibodies to theE ₂ glycoprotein of semilikiforest virus can protect mice from lethalencephalitis. J Gen Virol 1983;64:1405-1408.

[0005] Griffin D, Levine B, Tyor W, Ubol S, and Despres P, The role ofantibody in recovery from alphavirus encephalitis. Immunol Rev1997;159:155-161.

[0006] Hahn C S, Lustig S, Strauss E G, and Strauss J H, Western equineencephalitis virus is a recombinant virus. Proc Natl Acad Sci USA1988;85:5997-6001.

[0007] Harlow E and Lane D, Antibodies: A laboratory manual. Cold SpringHarbor Laboratory. Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1988.

[0008] Hayden M S, Gilliland L K, and Ledbetter J A, Antibodyengineering. Curr Opin Immunol 1997;9:201-212.

[0009] Hunt A R and Roehrig F T, Biochemical and biologicalcharacteristics of epitopes on the E1 glycoprotein of western equineencephalitis virus. Virology 1985;142:334-346.

[0010] Johnston R E and Peters C J, Alphaviruses. In: Fields Virology,3rd ed. Fields B N, Knipe D M, and Howley P M (Eds.). Raven Publishers,Philadelphia, 1996, pp. 843-898.

[0011] Laurino J P, Shi Q, and Ge J, Molecular antibodies, antigens, andmolecular diagnostics: a practical overview. Ann Clin Lab Sci 1999;29:158-166.

[0012] Long M C, Jager S, Mah D C W, JeBailey L, Mah M A, Masri S A, andNagata L P, Construction and characterization of a novel recombinantsingle chain variable fragment antibody against western equineencephalitis virus. Hybridoma 2000;19:1-13.

[0013] Mathews J H and Roehrig J T, Determination of the protectiveepitopes on the glycoproteins of Venezuelan equine encephalomyelitisvirus by passive transfer of monoclonal antibodies. J Inunol1982;129:2763-2767.

[0014] Netolitzky D L, Schmaltz F L, Rayner G A, Parker M D, Fisher G R,Bader D E, and Nagata L P, Complete genomic RNA sequence of westernequine encephalitis virus (strain 71 V-1658) and expression of thestructural genes. J Gen Virol 2000;81:151-159.

[0015] Rice S A, Long M C, Lam V, and Spencer C A, RNA polymerase II isaberrantly phosphorylated and localized to viral replicationcompartments following herpes simplex virus infection. J Virol1994;68:988-1001.

[0016] Schlesinger S and Schlesinger M J: Togaviridae, The viruses andtheir replication. In: Fields Virology, 3rd ed. Fields B N, Knipe D M,and Howley P M (Eds.). Raven Publishers, Philadelphia, 1996, pp.843-898.

[0017] Strauss J H and Strauss E G, The Alphaviruses: gene expression,replication, and evolution. Microbiol Rev 1994;58:491-562.

[0018] Strauss J H, Strauss E G, and Kuhn R J, Budding of alphaviruses.Trends Microbiol 1995; 3:346-350.

[0019] Verma R, Boleti E, and George A J T, Antibody engineering:Comparison of bacterial, yeast, insect, and mammalian expressionsystems. J Immunol Methods 1998;216:165-181.

[0020] Winter G and Milstein C, Man-made antibodies. Nature 1991;349:293-299.

[0021] Wright A, Shin S-U, and Morrison S L, Genetically engineeredantibodies. Crit Rev Immunol 1992; 12:125-168.

[0022] Xu B, Kriangkum J, Nagata L P, Fulton R E, and Suresh M R,Generation and characterization of a single chain Fv specific againstwestern equine encephalitis virus. Hybridoma 1999; 18:315-323.

[0023] Yamamoto K, Properties of monospecific antibodies to theglycoprotein of western equine encephalitis virus. Microbiol Immunol1986;30:343-351.

[0024] Yamamoto K, Hashimoto K, Chiba J, and Simizu B, Properties ofmonoclonal antibodies against glycoproteins of western equineencephalitis virus. J Virol 1985; 55:840-842.

[0025] Western equine encephalitis virus (WEE) is an envelopedpositive-sense, single-stranded RNA virus belonging to the alphavirusgenus. The 12 kb genome of WEE encodes for nonstructural (5′ end) andstructural (3′ end) proteins. The structural proteins are translatedfrom a subgenomic mRNA (26S mRNA) as a polyprotein that is processed byviral and cellular proteases into E1 (53 kDa), E2 (47 kDa), nucleocapsid[NC] (30 kDa), E3 (10 kDa), and 6K (6 kDa) proteins. The E1 and E2proteins are glycoproteins present in the lipid envelope. The E3 proteinis also a glycoprotein that is most often not a component of the virion,but is required for infectivity in wild-type virus. The NC proteinencloses the RNA genome in an icosahedral structure. The 6K protein isvirion associated and promotes efficient virus assembly (reviewed inStrauss and Strauss, 1994; Strauss et al., 1995; Johnston and Peters,1996; Schlesinger and Schlesinger, 1996).

[0026] WEE is localized to the Western hemisphere and poses a serioushazard to human health. Virus transmission is by infected mosquitoes,causing disease in humans and horses. Symptoms of WEE infection inhumans include encephalitis, convulsions, paralysis, malaise, fever,headaches, nausea, and vomiting. The case fatality rate in humans is 2%to 7%. Currently, there are no known antiviral drugs effective againstWEE. Although inactivated WEE vaccine exist for use in limitedpopulations such as laboratory personnel who are at high risk ofexposure to the virus, the immunogenicity of the inactivated WEE vaccineis often poor and the immunity is short-lived. Better protection againstWEE is required (Johnston and Peters, 1996).

[0027] Alphavirus antigenic properties and antibody neutralization havebeen studied with anti-alphavirus antibodies from mouse immunoglobulins.Murine antibodies capable of neutralizing virus have been generatedagainst E1 and E2 (Mathews and Roehrig, 1982; Boere et al., 1983;Yamamoto et al., 1985; Yamamoto, 1986). Mice were protected fromchallenge with WEE and Venezuelan equine encephalitis virus (VEE) wheninjected with antibodies against E1 and E2 in passive immunizationstudies (Mathews and Roehrig, 1982; Hunt and Roehrig, 1985; Yamamoto,1986). Anti-E2 monoclonal antibodies were able to protect mice fromlethal injections of Semliki Forest virus (SFV) (Boere et al., 1983).Furthermore, neutralizing and non-neutralizing antibodies to E1 and E2administered to mice, before or after infection with virus, wereprotected from Sindbis virus (SIN) (Griffin et al., 1997).

[0028] Animal antisera and monoclonal antibodies provide importantsources of antibody. Although recombinant antibodies have the advantagesof being produced quickly, economically, and in large quantities (Wrightet al., 1992; Hayden et al., 1997; Verma et al., 1998), recombinantantibodies grown in bacterial systems are often improperly folded andnonglycosylated (Wright et al., 1992; Verma et al., 1998). One may favorthe use of monoclonal antibodies over recombinant antibodies for avariety of reasons. Hybridoma technology is able to provide a wide rangeof monoclonal antibodies that bind to different antigens with highspecificity and affinity (Winter and Milstein, 1991; Laurino et al.,1999). Furthermore, monoclonal antibodies can be isolated with highpurity (Winter and Milstein, 1991; Laurino et al., 1999). Accordingly,production of monoclonal antibodies directed against WEE is desirable.

[0029] Up until recently, only a limited number of monoclonal antibodiesagainst WEE existd and have not been fully characterized. For instance,monoclonal antibodies produced by Hunt and Roehrig (1985) are capable ofimmunoprecipitating the E1/E2 heterodimer, identifying antigenicdeterminants on E1, and protecting mice when challenged with WEE.Monoclonal antibodies produced by Yamamoto et al. (1985), showingspecificity for E1 and E2 in enzyme-linked immunosorbent assays (ELISA),demonstrate neutralizing activity and are found effective in passiveimmunization studies (Yamamoto et al., 1985; Yamamoto, 1986). Recently,there have been studies directed to specific recombinant antibodiesagainst WEE. For example, Xu et al. (1999) successfully cloned ananti-WEE scFV. In addition, use of recombinant antibodies tohistologically stain the cells expressing WEE antigens was reported inNetolitzky et al. (2000). Accordingly, it is advantageous to produce andcharacterize a group of monoclonal antibodies for use in detecting anddiagnosing WEE effectively. It is also advantageous to study theinteractions between monoclonal antibodies with other alphaviruses, suchas VEE and SIN.

SUMMARY OF THE INVENTION

[0030] The present invention is directed to the construction andcharacterization of a group of mouse monoclonal antibodies against WEE.

[0031] An object of the present invention is to produce and identifyspecific monoclonal antibodies displaying various immunologicalactivities against WEE.

[0032] Another object of the present invention is to construct andcharacterize monoclonal antibodies capable of cross-binding to multiplealphaviruses.

[0033] It is another object of the present invention to manufacturerecombinant antibodies for hydridoma clones expressing anti-WEEmonoclonal antibodies.

[0034] It is yet a further object of the present invention to use theidentified monoclonal antibodies for immunodetection and immunotherpy.

[0035] According to one aspect of the present invention, it providesmonoclonal antibodies against WEE expressed from hybridomas.

[0036] According to another aspect of the present invention, it providesfor the construction of recombinant monoclonal antibodies from hybridomaclones against WEE, consisting of the steps of immunizing mice withantigens prepared from WEE infected cells; fusing and cloning hydridomacells lines from the immunized mice; and genetic engineering recombinantantibodies from said cultured hybridoma cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037]FIG. 1. WEE indirect ELISA with increasing amounts of antigen.Varying amounts of inactivated WEE antigen were immobilized onto 96 wellplates, after which 20 μg/ml purified 3F3E9G5 antibody was added to eachof the wells. Binding was detected with horseradishperoxidase-conjugated antibodies and ABTS solution. The plates were readat an absorbance of 405 nm.

[0038]FIG. 2. Western blot analyses with WEE antibodies. Inactivated WEEantigen was run on SDS-PAGE gels (12%) and immunoblotted. The sampleswere probed with various WEE antibodies. Lanes: (1) 2D1E11F8; (2)11H9E2C12; (3) 9B10D4D11G4; (4) 10A7D10F5; (5) 3F3E9G5; (6) 3F6E3F8; (7)1G6C1H5; (8) 10B5E7E2; (9) 11D2E11F2; (10) 5F1F2G1.

[0039]FIG. 3. Immunoprecipitation of WEE proteins. In vitro translatedWEE proteins were synthesized from pCXH-3 and rabbit reticulocyte lysatein the presence of [³⁵S]-methionine. Radiolabeled proteins wereimmunoprecipitated with antibodies and protein G-agarose, run onSDS-PAGE gels (12%), and analyzed by autoradiography. Lanes: (1) MWmarkers; (2) in vitro synthesized WEE proteins; (3) 10B5E7E2; (4)10A7D10F5; (5)11H9E2C12; (6) no antibody control; (7) 3F3E9G5; (8)8F8D2F7E11; (9) 5C5A1H11.

DETAILED DESCRIPTION OF THE INVENTION

[0040] Materials and Methods

[0041] Preparation of Mouse Monoclonal Hybridoma Cell Lines

[0042] Mice (BALB/c, Charles River) were immunized with three doses of20 μg of gradient purified, formalin inactivated antigen prepared fromWEE strain B11 infected Vero cells (CCL-81, American Type CultureCollection, Manassa, Va.), as previously described (Xu et al., 1999;Long et al., 2000), and 50 μL TiterMax® (CytRx Corp., Norcross, Ga.)adjuvant in a total volume of 100 μl. The injections were givenintraperitoneally at three week intervals. Three weeks after the thirdinjection, the mice were given 10 μg of inactivated WEE antigenintravenously, in a total volume of 50 μl in phosphate-buffered saline(PBS). The fusions were performed on spleen cells 5-7 days later.Fusions, initial screening, and subcloning were performed by theHybridoma Facility, Southern Alberta Cancer Research Centre, Universityof Calgary, Calgary, Alberta. Hybridoma cell lines were grown andmaintained in RPMI 1640 media supplemented with 10% heat-inactivatedfetal calf serum, 2 mM L-glutamine, 1× vitamins solution,antibiotic/antimycotic solution (100 units/ml penicillin G, 100 μg/mlstreptomycin, and 25 μg/ml amphotericin B), 100 μM nonessential aminoacids, and 1 mM sodium pyruvate. All tissue culture reagents werepurchased from Gibco BRL, Gaithersburg, Md. The hybridoma cells weremaintained at a density of 0.5-1.0×10⁶ cells/ml and doubledapproximately every 24 hr.

[0043] Purification of Antibodies

[0044] Various hybridoma clones producing anti-WEE antibodies (3F3E9G5,5C5A1H11, 9B10D4D11 G4, 10B5E7E2, 11H9E2C12) were cultured in growthmedia in T150 flasks. Media supernatants were collected at 24 hr timepoints and used as starting material for antibody purification. Thesupernatants were passed over protein G columns (Pierce, Rockford, L),which were subsequently washed with ImmunoPure® binding buffer (Pierce).Bound IgG was eluted with ImmunoPure® elution buffer (Pierce) and six 1ml fractions were collected. All fractions were neutralized with 100 μlof 1 M Tris-HCl pH 7.5 and monitored by absorbance at 280 nm. Allantibodies eluted in fractions 3 and 4. The eluted antibodies werefurther desalted using Excellulose™ columns (Pierce) equilibrated in PBSpH 7.5. Concentrations of antibodies eluted were determined byabsorbance (280 nm) measurements (1 mg/ml IgG=A280/1.44).

[0045] WEE Indirect ELISA

[0046] The WEE indirect enzyme-linked immunosorbent assay (ELISA) wasperformed as described by Long et al., 1999. In brief, inactivatedantigen from WEE strain B11 infected Vero CCL-81 cells was prepared byprevious methods (Xu et al., 1999; Long et al., 2000). Varyingconcentrations of inactivated WEE antigen or BSA were immobilized ontoNunc Maxisorp™ flat bottomed 96 well plates (Gibco BRL). The wells wereblocked, washed, and then incubated with mouse monoclonal antibodies for1 hr at 37° C. The antibodies were diluted to various concentrations inwash buffer consisting of PBS, 0.05% Tween 20, and 0.1% BSA. The wellswere subsequently washed and incubated for 1 hr at 37° C. with secondaryantibody, horseradish peroxidase-conjugated goat anti-mouse antibody, ata 1:3,000 dilution in wash buffer. The plates were washed and incubatedwith a 1:1 solution of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) and hydrogen peroxide (Kirkegaard and PerryLaboratories, Inc., Gaithersburg, Md.). The plates were read at anabsorbance of 405 nm.

[0047] Western Blotting of WEE

[0048] Western blotting was performed as described previously (Rice etal., 1994; Long et al., 2000). In brief, 50 μg formalin inactivated WEEantigen (described above) was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). WEE proteins wereseparated on a 12% discontinuous polyacrylamide gel, after whichproteins were transferred from the gel to Immobilon™-P membranes (0.45μm pore size, PVDF filter type) (Millipore, Bedford, Mass.). The filterswere blocked, washed in PBS containing 0.1% Tween 20 and 0.02% SDS, andthen probed with anti-WEE mouse monoclonal antibodies for 1 hr at roomtemperature. The Mini-Protean® II multi-screen apparatus (Bio-RadLaboratories, Mississauga, Ontario) was used to analyze multipleantibody samples per immunoblot. The primary antibodies used weredifferent dilutions of culture supernatants or different concentrationsof purified protein in wash buffer. These blots were washed andincubated with a 1:3,000 dilution of horseradish peroxidase-conjugatedgoat anti-mouse immunoglobulin (H+L) (Caltag Laboratories, Burlingame,Calif.) for 1 hr at room temperature. Proteins were detected using theenhanced chemiluminescence (ECL) method (Amersham Pharmacia Biotech,Baie D'Urfe, Quebec).

[0049] Isotyping Antibodies

[0050] Isotyping of the antibodies was performed using the monoclonalantibody-based mouse immunoglobulin isotyping kit (Pharmingen,Mississauga, Ontario). Specific rat anti-mouse antibodies (IgG₁, IgG₂a,IgG₂b, IgG₃, IgM, IgA, Ig κ, and Ig λ) were coated onto Nunc Maxisorp™flat bottomed 96 well plates. The wells were washed with PBS containing0.05% Tween-20 and blocked with PBS containing 1% BSA for 30 min at roomtemperature. Supernatants of anti-WEE hybridoma cells or purifiedanti-WEE antibodies were then added to the wells and incubated for 1 hrat room temperature. The wells were subsequently washed and incubatedwith alkaline phosphatase-conjugated rat anti-mouse Ig antibody for 1 hrat room temperature. After washing, detection of the plates wasperformed by incubating the wells with phosphatase substrate solutionfor 30 min at 37° C. The isotypes of the antibodies were determined byidentifying positive yellow reactions, corresponding to specificantibody isotypes. In addition, antibody isotypes were confirmed usingalternate isotyping kits from Cedarlane Laboratories Ltd., Homby,Ontario and Gibco BRL.

[0051] Immunoprecipitation of WEE

[0052] WEE proteins were prepared in one-step in vitro transcription andtranslation reactions using the TNT® coupled system (PromegaCorporation, Madison, Wis.). In the presence of rabbit reticulocytelysate, transcription of the pCXH-3 plasmid resulted in WEE RNA. ThepCXH-3 plasmid was constructed by cloning the entire WEE 26S region intopCI (Promega) at the Xba I and Sma I restriction sites. The WEE 26Sregion was placed under the control of the T7 RNA polymerase promoterand the cytomegalovirus enhancer/promoter (Netolitzky et al., 2000). TheRNA was translated in the presence of [³⁵S]-methionine to produceradiolabeled WEE proteins, which were further processed with caninepancreatic microsomal membranes. All components of the in vitrotranscription and translation reactions were incubated together for 90min at 30° C.

[0053] The TNT® reactions were diluted to a volume of 500 μl with RIPbuffer consisting of 0.15 M sodium chloride, 0.1% SDS, 50 mM Tris-HCl pH7.4, and 1% Triton X-100, and subjected to a preabsorption step byincubating with 7511 of protein G-agarose (Gibco BRL) for 30 min at roomtemperature. The samples were centrifuged at 13,000×g for 1 min, and thesupernatants were then incubated with either 100 μl of supernatants fromanti-WEE hybridoma cells or 20 μg of purified anti-WEE antibodies. Thereactions were incubated for 1.5 hr at room temperature, after which 75μl of protein G-agarose was added. The reactions were incubated for anadditional 30 min at room temperature. Immunoprecipitated proteins werecollected by centrifuging at 13,000×g for 1 min. The pellets were washedwith 500 μl of RIP buffer and centrifuged at 13,000×g for 1 min; thisstep was repeated three additional times. The pellets were resuspendedin 2× Tricine sample buffer (Bio-Rad Laboratories) containing fresh 2%β-mercaptoethanol and heated at 100° C. for 10 min. The samples werecentrifuged at 13,000×g for 1 min, and the supernatants were collected.The immunoprecipitated [³⁵S]-labeled WEE proteins were further analyzedby SDS-PAGE and autoradiography. Radiolabeled [¹⁴C]-molecular weightmarkers from Amersham Pharmacia Biotech were also run on thepolyacrylamide gels.

[0054] Results

[0055] Preparation of Mouse Monoclonal Hybridoma Cell Lines andPurification of Antibodies

[0056] A total of 24 hybridoma cell lines which reacted with inactivatedWEE antigen in ELISA assays were isolated. Of the 24 cell lines, 17 werechosen for further in-depth analysis (Table 1). A select number ofanti-WEE antibodies (3F3E9G5, 3F6E3F8, 9B10D4D11G4, 10B5E7E2,8F8D2F7E11, 11D2E11F2, and 11H9E2C12) were purified using protein Gcolumns. In all purifications, concentrations of antibody were highestin fractions 3 and 4, ranging from 1 to 2 mg/ml.

[0057] Antigen Binding Activity of Antibodies

[0058] The present study first sought to determine the antigen bindingactivity of monoclonal antibodies using the indirect ELISA assay. WEEantigen (10 μg/ml) was immobilized onto 96 well plates and incubatedwith antibody. Absorbance values of controls, where no antigen waspresent, were subtracted from absorbance values for samples containingantigen. At various dilutions, each of the supernatants possessedantigen binding activity and displayed absorbance (405 nm)readings >0.154 (data not shown). The antigen binding activity of eachof the antibodies is compared in Table 1, where the maximum dilutions ofantibody supernatant used in ELISAs are listed. Certain antibodies(2B7C8G2, 3F3E9G5, 3F6E3F8, 5C5A5E5, and 10B5E7E2) showed strongreactivity at >{fraction (1/320)} dilutions, whereas other antibodies(1G6C1H5, 5C5B7H10, 5F11F2G11, and 10A7D10F5) showed weak reactivity toWEE at {fraction (1/20)} dilutions. Absorbance readings were also takenwith different antigen concentrations, at fixed concentrations of the3F3E9G5 antibody (20 μg/ml) (FIG. 1). Generally, increasingconcentrations of antigen resulted in gradual increasing absorbancevalues or antibody-antigen binding. At a concentration of 20 μg/mlantibody, the antibodies displayed a lower limit of detection of <1μg/ml antigen. The ELISA data showed that the mouse monoclonalantibodies were functionally active, as demonstrated by their ability tobind to WEE antigen.

[0059] The study next sought to determine which WEE proteins werespecifically recognized by each of the mouse monoclonal antibodies.Western blotting techniques found that a subset of antibodies werecapable of detecting WEE proteins (FIG. 2). The antibodies 1G6C1H5,3F6E3F8, SF11F2G11, 10A7D10F5, and 11D2E11F2 recognized E1 atapproximately 53 kDa (FIG. 2, lanes 7, 6, 10, 4, and 9 respectively),whereas the antibodies 3F3E9G5, 9B10D4D11G4, and 10B5E7E2 recognized E2at approximately 47 kDa (FIG. 2, lanes 5, 3, and 8 respectively). It isimportant to note that it is difficult to distinguish between E1 and E2,as both proteins often comigrate on 12% polyacrylamide gels. The 30 kDanucleocapsid (NC) is recognized by 2D1E11 F8 and 11H9E2C12 (FIG. 2,lanes 1 and 2 respectively). WEE proteins were not detected by theantibodies 2B7C8G2, 2H1D12E2, 5C5A1H11, 5C5A5E5, 5C5B7H10, 5C5C7C4, and8F8D2F7E11 by Western blotting, although WEE proteins were detected bythese particular antibodies in ELISAs. This may be because the ELISA ismore sensitive than the Western blot. Furthermore, antibodies detected“native” WEE proteins in an ELISA, as opposed to “denatured” WEEproteins in a Western blot. To further characterize each of the mousemonoclonal antibodies, the isotypes of the antibodies were determined(Table 1). The antibodies displayed IgG₁, IgG₂a, or IgG₂b isotypes; noneof the antibodies showed, IgG₃, IgA, and IgM isotypes. Most of theantibodies were either IgG₁ or IgG₂a, with kappa light chains. Oneantibody, 11H9E2C12, was IgG_(2b) with lambda light chains. Theisotyping data provided information for immunoprecipitation experiments.Protein G was used to immunoprecipitate the antibody-antigen complexessince protein A does not have strong affinity for the mouse IgG₁subclass (Harlow and Lane, 1988). For antibodies displaying IgG₂a andIgG_(2b) isotypes, additional immunoprecipitation experiments were donewith protein A.

[0060] Immunoprecipitation experiments were performed to demonstratethat the antibodies were capable of binding to native WEE proteins. Itwas found that only a limited number of antibodies were capable ofimmunoprecipitating WEE proteins with protein G agarose (FIG. 3). Invitro translated E1, E2, and NC were first produced from the TNT® system(FIG. 3, lane 2). The bands for E1 and E2 run together. Furthermore, theNC runs as a broad band. The antibodies 10A7D10F5 and 8F8D2F7E1 weaklyimmunoprecipitated E1 (FIG. 3, lanes 4 and 8 respectively), whereas theantibodies 3F3E9G5 and 5C5A1H11 immunoprecipitated E2 (FIG. 3, lanes 7and 9 respectively). Lastly, 11H9E2C12 immunoprecipitated the NC (FIG.3, lane 5). No WEE proteins were immunoprecipitated with 10B5E7E2 (FIG.3, lane 3), in the absence of antibody (FIG. 3, lane 6), or with proteinA (data not shown). All other antibodies did not show anyimmunoprecipitation activity, although they displayed ELISA activity.This may be due to immunoprecipitations being less sensitive thanELISAs. Interestingly, 5C5A1H11 and 8F8D2F7E11 were able toimmunoprecipitate WEE proteins but not able to detect WEE proteins byWestern blotting. The antibodies may be capable of detecting WEEproteins in proper conformation in an immunoprecipitation but not WEEproteins as linear epitopes in Western blotting. Certain antibodies,3F3E9G5, 10A7D10F5, and 11H9E2C12 were able to recognize both “native”and “denatured” proteins as they showed positive reactivity to WEEantigen in ELISA, Western blotting, and immunoprecipitations.

[0061] Lastly, cross-reactivity experiments were performed in order todetermine if the anti-WEE antibodies displayed binding activity to otheralphaviruses. Antigen from SIN, VEE, or EEE was immobilized onto ELISAplates and incubated with antibody. Several antibodies, 3F3E9G5,9B10D4D11G4, and 11D2E11F2, cross-reacted with SIN antigen, whereasother antibodies 2B7C8G2, 2D1E11F8, 5C5A1H11, and 11H9E2C12 reacted withEEE (Table 1). The 11H9E2C12 antibody bound not only to WEE and EEE, butalso VEE.

[0062] Discussion

[0063] Protection from WEE infection and disease are relevant andimportant issues affecting a large number of the population. It has beenfound that in mice, protection from alphavirus by activated T cellsalone is not effective or sufficient. Instead, clearance and protectionfrom infectious virus in the nervous system are accomplished bydelivered antibodies (Griffin et al., 1997). Thus, an important methodfor protection against WEE may be facilitated by passive immunization,where viral-specific antibodies are administered to help prevent illnessor mediate recovery of individuals exposed to virus. A limited number ofmonoclonal antibodies with both neutralization and passive immunizationactivity against alphaviruses have been previously found (Mathews andRoehrig, 1982; Boere et al., 1983; Yamamoto, 1986; Johnston and Peters,1996; Griffin et al., 1997). The present study reports the constructionand characterization of a collection of mouse monoclonal antibodiescapable of recognizing WEE proteins for potential use in identificationand therapy studies.

[0064] The 24 hybridomas expressing different anti-WEE antibodies areisolated, and the most reactive antibodies are evaluated further. Themonoclonal antibodies all show varying reactivity to WEE in ELISAs, bothwith the supernatants and with the purified fractions. The antibodies2B7C8G2, 3F3E9G5, 3F6E3F8, 5C5A5E5, and 10B5E7E2, display the highestbinding activity to WEE, at dilutions >{fraction (1/320)}. Othersdisplay binding activity at dilutions >{fraction (1/100)}. Detection ofWEE antigen by 3F3E9G5 is sensitive to less than 1 μg/ml when 20 μg/mlof antibody is used. These antibodies are strong candidates for use inELISA based WEE detection assays. Of these antibodies listed, only3F3E9G5 (E2), 3F6E3F8 (E1), and 10B5E7E2 (E2) are reactive against WEEprotein in Western blotting. Furthermore, of these three antibodies,only 3F3E9G5 is reactive in immunoprecipitations. From these results, itappears that of the strongly binding WEE antibodies, 3F3E9G5 is the mostversatile antibody, capable of recognizing WEE proteins in “native” and“denatured” forms and in a number of different assays.

[0065] Many of the antibodies, 1G6C1H5 (E1), 2D1E11F8 (NC), 3F3E9G5(E2), 3F6E3F8 (E1), 5F11F2G11 (E1), 9B10D4D11G4 (E2), 10A7D10F5 (E1),10B5E7E2 (E2), 11D2E11F2 (E1), and 11H9E2C12 (NC) display activity inWestern blotting and recognize WEE proteins with clear resolution. Theantibodies 3F3E9G5, 10A7D10F5, and 10H9E2C12 are not only capable ofrecognizing WEE proteins in Western blotting but also inimmunoprecipitations, indicating that these three antibodies may becapable of recognizing E2, E1, and NC respectively in not only“denatured” but also “native” forms. 5C5A1H11 (E2) and 8F8D2F7E11 (E1)mayonlyrecognize WEE proteins in their “native” forms, as theseantibodies are reactive only in ELISAs and immunoprecipitations but notin Western blotting.

[0066] The monoclonal antibody against western equine encephalitisvirus, reference no. 8F8D2F7E11, has been deposited at the InternationalDepositary Authority of Canada, National Microbiology Laboratory, HealthCanada, at 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3R2 onJan. 13, 2004 and was assigned Accession No. 120104-01.

[0067] A subgroup of the anti-WEE antibodies is also capable of bindingto other alphavirus antigens. The antibodies 3F3E9G5, 9B10D4D11G4, and11D2E11F2 bind to SIN, whereas 2B7C8G2, 2D1E11F8, and 5C5A1H11 bind toEEE. One antibody, 11H9E2C12, recognizes three different alphaviruses,WEE, VEE, and EEE. This is not entirely surprising as a large number ofviruses in the alphavirus genus are closely related in terms ofmolecular characteristics and structure (Strauss and Strauss, 1994;Johnston and Peters, 1996). For instance, alphavirus nucleocapsids areantigenically similar. The nuclecapsid gene of WEE is closely related tothe analogous regions of EEE (Hahn et al., 1988). Interestingly,2D1E11F8 and 11H9E2C12, antibodies which recognize the nucleocapsid ofWEE, bind to EEE. The E1 and E2 sequences of WEE are most closelyaligned with comparable sequences of SIN (Hahn et al., 1988). This studyalso finds that 3F3E9G5, 9B10D4D11G4, and 11D2E11F2 bind to one of theWEE glycoproteins and is cross-reactive with SIN. Because the aboveantibodies recognize other alphaviruses in addition to WEE, theantibodies may potentially have functions in multiple systems, not onlyin WEE based assays but also in SIN, VEE, and EEE immunodetections andimmunotherapies.

[0068] The information derived from the characterization of the mousemonoclonal antibodies may be used in further immunological studies.These antibodies can be used to detect WEE in a number of forms, as theantibodies have different specificities and reactivities in variousassays. Recombinant antibodies such as scFv, Fab, and bispecificantibodies, can be constructed from each of the hybridoma clonesexpressing anti-WEE monoclonal antibodies. From the hybridoma expressing10B5E7E2, a scFv retaining good recognition to the WEE antigen wasconstructed. This scFv was fused to the human IgG₁ heavy chain toproduce a chimeric antibody which may show potential for immunotherapy(Long et al., 2000). These recombinant antibodies along with the mousemonoclonal antibodies can serve in a wide range of applications, rangingfrom immunohistochemistry immunoassays, radioimmunodiagnosis,radioimmunotherapy, and immunotherapy (Hayden et al., 1997; Cao andSuresh, 1998).

[0069] It is to be understood that the embodiments and variations shownand described herein are merely illustrative of the principles of thisinvention and that various modifications may be implemented by thoseskilled in the art without departing from the scope and spirit of theinvention. TABLE 1 Monoclonal antibody specificities, isotypes, andcross-reactivities WESTERN AND IMMUNO- PRECIPITATION IsotypingCrossreactivity ANTIBODY SPECIFICITY ELISA FUSION PARTNER IgG₁ IgG_(2a)IgG_(2b) Kappa Lambda SIN VEE EEE 1G6C1H5 E1  1/20 Sp2/mIL-6 + + 2B7C8G2— >1/320 P3/NSI/1Ag-1-NSI + + + 2D1E11F8 NC  1/80 P3/NSI/1Ag-1-NSI + + +2H1D12E2 —  1/40 P3/NSI/1Ag-1-NSI + + 3F3E9G5 E2 >1/320 Sp2/mIL-6 + + +3F6E3F8 E1 >1/320 Sp2/mIL-6 + + 5C5A1H11 E2  1/160P3/NSI/1Ag-1-NSI + + + 5C5A5E5 — >1/320 P3/NSI/1Ag-1-NSI + + 5C5B7H10 — 1/20 P3/NSI/1Ag-1-NSI + + 5C5C7C4 —  1/40 P3/NSI/1Ag-1-NSI + +5F11F2G11 E1  1/20 P3/NSI/1Ag-1-NSI + + 8F8D2F7E11 E1  1/160Sp2/mIL-6 + + 9B10D4D11 E2  1/160 P3/NSI/1Ag-1-NSI + + + 10A7D10F5 E1<1/20 Sp2/mIL-6 + + 10B5E7E2 E2 >1/320 Sp2/mIL-6 + + 11D2E11F2 E1  1/80Sp2/mIL-6 + + + 11H9 NC  1/160 P3/NSI/1Ag-1-NSI + + + +

1-25. (Canceled).
 26. A method comprising binding a monoclonal antibody(Mab) specifically to the western equine encephalitis E1 glycoprotein,wherein said Mab is expressed from a hybridoma, and wherein saidhybridoma is deposited with the International Depositary Authority ofCanada under Accession Number 120104-01.
 27. The method of claim 26,wherein the specific binding of said Mab to the western equineencephalitis E1 glycoprotein is indicative of western equineencephalitis virus infection.
 28. The method of claim 26, wherein thespecific binding of said Mab to the western equine encephalitis E1glycoprotein is the mechanism of action in a treatment for westernequine encephalitis virus infection.